In the male, follicle stimulating hormone (FSH) binds to G-protein coupled transmembrane receptors on the Sertoli cells and controls their development and function. Sertoli cells are the somatic cells of the testis that provide physical scaffolding and various signaling factors to the proliferating and differentiating germ cells. During the mouse testis development, Sertoli cells rapidly divide up to postnatal day 14, thereafter cease to proliferate and terminally differentiate. The number of Sertoli cells determines the germ cell capacity and thus the male reproductive potential. Aberrations in Sertoli cell proliferation and differentiation may cause male infertility. Despite this knowledge, the molecular basis for FSH regulation of Sertoli cell proliferation and differentiation is unknown. A major challenge to study the mechanism of FSH action in the male has been to rapidly isolate pure Sertoli cells (free of contaminating testis cell types) that accurately reflect their in vivo function. The long-term goal of this project is to understand how FSH regulates distinct developmental programs in Sertoli cells by orchestrating various gene/protein networks. In Specific Aim 1, we will isolate pure populations of Sertoli cells by two approaches. In the first approach, a rapid magnetic cell separation method will be used to selectively enrich Sertoli cells based on their unique cell surface expression of FSH-receptors. In the second approach, Sertoli cells will be enriched from two different strains of transgenic mice expressing an enhanced green fluorescent protein (EGFP) marker (driven by either Mullerian inhibiting substance or Pem homoebox promoter) specifically in the Sertoli cell lineage. Additionally, by crossbreeding, the GFP transgenes will be introduced into the FSH-null background. Pure populations of Sertoli cells will be isolated from both these strains of mice (with and without FSH) by flow cytometry. In Specific Aim 2, to begin to identify FSH-responsive genes, we will use subtractive hybridization strategies to enrich Sertoli cell-specific cDNAs or use cDNA microarrays to analyze large-scale gene expression changes in pure populations of Sertoli cells (isolated in Specific Aim 1) during proliferation (day 10) and differentiation (day 42) phases. Collectively, these experiments will enable us to monitor the development of GFP-expressing Sertoli cells in vivo, develop rapid methods to purify Sertoli cells and identify and characterize the FSH-responsive genes in them. Finally, the GFP transgenic mice and the Sertoli cell - specific cDNAs and large-scale gene expression profiles generated in this pilot grant will be valuable reagents and will be made available to a number of investigators engaged in male reproductive endocrinology and developmental biology research.